The generation of libraries of small molecules and selection of those molecules that bind uniquely to a target of interest is important for drug discovery. The production of genetically-encoded libraries, in which each library member is linked to an information template, such as DNA or RNA, makes it possible to process large chemical libraries without separating individual library members into individual solutions and reaction vessels. One can select target molecules from mixtures of genetically-encoded molecules and identify or amplify the selected molecule of interest using its information template.
Phage display is one example of a genetically-encoded library. Phage display is a well known technique used in the analysis, display and production of protein antigens, especially human proteins of interest. Phage display is a process during which the phage, a bacterial virus, is made to expose or “display” different peptides or proteins including human antibodies on its surface. Through genetic engineering, peptides or proteins of interest are attached individually to a phage cell surface protein molecule (usually Gene III protein, g3p). In such a phage population (phage library), each phage carries a gene for a different peptide or protein—g3p fusion and exposes it on its surface. Through a variety of selection procedures, phages that “display” binders to specific target molecules of interest can be identified and isolated. These binders can include interaction partners of a protein to determine new functions or mechanisms of function of that protein, peptides that recognize and bind to antigens (for use in diagnosis and therapeutic targeting, for example), and proteins involved in protein-DNA interactions (for example, novel transcription factors).
The phage display technique can be very useful in discovery and development of pharmaceutical and/or diagnostic products. In phage display the entire phage binds and can be eluted from an immobilized target molecule. Since the phage remains infective it can inject its DNA into bacterial cells and is amplified. The main limitation of phage display, however, is the occurrence of non-specific adsorption of phages during the binding stage, which necessitates enrichment over several rounds and individually tailored washing and elution conditions. Phage display methods are usually restricted to the production of libraries, which can be encoded by direct DNA-RNA-protein information transfer. These methods are typically limited to linear sequences of peptides, made of only 20 natural amino acids.
Typically, the amplification of libraries of peptides on the surface of the phage requires an in vitro translation system, in which DNA is modified to express the displayed peptides of interest. The generation and use of such translation systems can be expensive and time consuming. The use of self-replicating species such as phage or bacteria simplifies amplification of libraries because each library member is amplified “spontaneously”, when given the appropriate resources. For example, for phage displayed libraries, adding one phage to a simple culture broth with bacteria can produce an arbitrarily large population of phage for a very low cost.
Several methods exist which involve conversion of libraries of phage-displayed polypeptides to libraries of peptide derivatives. Typically, these methods use organic synthesis on the peptides to make peptide derivatives. The characterization and improvement of reaction yields is an important cornerstone of organic synthesis. Bulk biochemical methods, such as western blot and mass spectrometry, are often used, to quantify the amount of product obtained or to determine the success of generating the desired reaction products. However, in the absence of this characterization, the synthesis cannot be claimed to be reliable or reproducible. Reactions used for synthesis of such libraries of peptide derivatives have typically been validated using one phage clone or one purified peptide. The actual synthesis of libraries is typically done “blindly”, and the efficiency of such synthesis is unknown. The quality of the libraries generated by this method is, thus, usually unknown. While selection from these libraries can provide useful non-peptidic molecules, overall the efficiency of such selection is unclear.
Jesper at al. in U.S. Pat. No. 6,017,732, describe chemical modification of point residues in antibodies displayed on phagemid. This patent discusses a limited set of reactions including the alkylation of a Cys residue within an antibody, and subsequent detection with fluorescence and radioactive probes. The technique described in Jesper is designed for estimating the yield in large (>1010 copies) clonal populations of phage. However, there is no teaching or suggestion of the quantification or optimization of a derivative library synthesis.
U.S. Pat. No. 7,141,366 to Noren et al, describes the production of phage with a unique chemical residue, selenocysteine (Sec), incorporated in a specific location in a phage-displayed peptide. Phage-displayed peptide libraries that contain Sec were generated, thus creating modified peptide libraries. Bulk biochemical methods, such as western blot, were used to qualitatively characterize the chemical modifications that occur on the Sec residue. However, this method cannot be extended to Sec-free peptide libraries, and, further, there is no teaching or suggestion of the quantification or optimization of a derivative library synthesis.
US Patent Publication 2009/0137424 to Schultz et al, describes the production of phage with non-natural aminoacids incorporated at a specific location within a phage-displayed peptide using orthogonal tRNA and aminoacyl tRNA synthase. Phage-displayed peptide libraries that contain azido phenyl alanine (AzPhe) are generated. Bulk biochemical methods, such as western blot and mass spectrometry, are used to qualitatively characterize the chemical modifications that occur on AzPhe residues. No methods for quantification of chemical yields in single or multi-step reactions in the synthesis of the library are described, nor strategies for improvement of chemical yields.
U.S. Pat. No. 6,642,014 to Pedersen et al, describes the use of capture agents to select enzymes with improved or new activities. This patent discusses the generation of chemicals by enzyme catalysis, not by direct chemical transformation. Specially-engineered phagemid is used and required to display both enzyme and substrate on the surface of phage.
US Patent Publication 2010/0317547 to Winter and Heinis, describes methods for the generation of a library of bicyclic peptides displayed on phage from a linear peptide, which contains random amino acids flanked by three cysteine residues.
Typically, and as indicated in the above references, the phage display methods known in the art are not designed for efficiency and success of the reactions during synthesis. These previous systems are often characterized on purified protein from phage well after the derivatizing reaction has taken place. The yields of reactions on phage, and the quality and purity of the library are, thus, generally unknown.
Thus, there remains a need for quantitative characterization of peptide-derivative phage display to determine the quality of the phage display procedure and a corresponding optimization of the procedure to ensure optimal yields of reaction products.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present application. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.